This proposal describes a five-year training program for the development of an academic career in Cardiology. The principle investigator obtained his MD/PhD from Stanford School of Medicine and has completed clinical training in Internal Medicine, Cardiology, and Interventional Cardiology at Massachusetts General Hospital (MGH). He will now focus on the study of stem cell and developmental biology as it pertains to cardiac disease under the mentorship of Dr. Kenneth Chien. Dr. Chien, a leader in the field of cardiac development, is chief of the Cardiovascular Research Center (CVRC) at the MGH and Leader of the Cardiovascular Disease Program at the Harvard Stem Cell Institute (HSCI). He has trained numerous physician scientists. To promote training in stem cell biology, the principle investigator has already spent one year training with Dr. Stuart Orkin, a pioneer in stem cell biology. Dr. Orkin will continue to play a prominent role in training the principle investigator as an Advisory Committee member. In addition, Dr. Kenneth Bloch and Dr. Randall Peterson, both recognized leaders in cardiovascular biology, will serve mentorship roles on the Advisory Committee. The CVRC and the HSCI provide an ideal setting for training physician- scientists in the rapidly converging areas of cardiac development and stem cell biology. The research program will focus on lineage commitment and differentiation during murine cardiac development. Acquired and congenital heart disease represents a leading cause of mortality and morbidity in the world and many of the genes important in cardiac development have been implicated in human congenital heart disease. Defining the molecular pathways that control cardiac development is essential to understanding the pathophysiologic basis of these diseases and will contribute to the scientific basis of regenerative medicine. Recently, small non-coding RNA molecules called microRNAs (miRNAs) have been shown to regulate gene expression by titrating the dosage of critical proteins. Although several miRNAs are enriched in the developing heart and appear to control the balance of myocardial expansion and differentiation, the full temporal and spatial repertoire of cardiac miRNAs has yet to be established. The mammalian heart develops from two closely related sets of cardiac progenitors, first heart field (FHF) which gives rise to the left ventricle (LV) and the second heart field (SHF) which gives rise to the right ventricle (RV) and outflow tract (OFT). To investigate the role of miRNAs in controlling the development of these lineages, we have used distinct genetic markers to develop a two-color transgenic murine system. We were thereby able to purify FHF and SHF progenitors from embryos and embryonic stem cells differentiating in vitro, and to identify a set of miRNAs that are differentially expressed in the two cardiac lineages. Significantly we identify miR200a and miR200b (miR200), two closely related miRNAs transcribed in a polycistronic fashion, as the first miRNAs specific for early FHF progenitors within the developing heart and in preliminary experiments demonstrate that they appear to play a key role in the normal development of that lineage. We hypothesize that miR200 regulates cardiac development by promoting FHF progenitor expansion and differentiation and suppressing commitment to other lineages. We further hypothesize that miR200 functions by controlling specific target genes and is necessary for normal morphogenesis and function of the mature mammalian heart. To test this, we propose the following specific aims: 1) Determine if miR200 is promotes FHF development by supporting mesoderm differentiation and suppressing endoderm and ectoderm differentiation; 2) Determine the mechanisms by which miR200 controls FHF development; 3) Determine the role of miR200 during embryonic development. The tools and understanding developed here will contribute to the scientific basis of cardiac regenerative therapies. PUBLIC HEALTH RELEVANCE: Heart disease affects millions of Americans every year and is a leading cause of hospitalization in the United States. Understanding how the heart develops will help us understand how the heart becomes diseased. This study will give us clues for the future on how to diagnose and treat cardiac birth defects and adult diseases.